The mammalian heart is well supplied by autonomic nerves that harmonize the cardiac rhythmic activities. Recently, at least seven neuropeptides have been found to occur in the nerves of the heart. In a current NIH-funded study, the Principal Investigator has mapped the peptide-containing nerves in the heart and classified the cardiac innervation into 3 major divisions based on their origins. The first intrinsic nerve type has its origin in the Type I intrinsic ganglia and contains neuropeptide Y. The second has dual origins from both Type II intrinsic ganglion cells and the paracardiac ganglion cells and contains catecholaminergic neurotransmitters. The third type has its origin entirely from outside of the heart and contains other neurochemicals including most of the peptides. The picture of cardiac innervation emerging from this study is so different from the classic view that a detailed study of the ganglionic origin and the entire pathway of each neurochemical nerve type is warranted. We propose to study the organization and the origin of the cardiac innervation of rat and guinea pig by retrograde and anterograde labelling techniques in combination with immunocytochemistry. True blue, horseradish peroxidase (HRP) , and wheat germ agglutinin-HRP conjugate will be used as the tracing reagents. They will be injected into different regions of the heart and the spinal cord. Once the neuronal pathway tracers are detected in neurons or nerves at a distant site, a panel of 12 antibodies will be applied on the adjacent tissue sections with immunocytochemistry to identify classic and peptide neurochemicals contained in the cells. When two or more types of immunoreactivities are identified in the same nerves, conventional and double immunogold electron microscopy will be performed on tissues taken from the same region to identify the characteristics of possible co-storage of the neurotransmitters. In situ hybridization will also be performed to study the neuropeptide gene expression from these ganglia. The results obtained from this study will provide a detailed morphologic and neuroanatomic foundation for a better understanding of the functional significance of neuropeptides in the cardiac innervation.